瘦素/ERK信号在云锡矿粉诱导大鼠II型肺泡上皮细胞转化中的作用

背景与目的 目前，云南个旧锡矿有大量矿工从事开采工作，这种职业环境与接触粉尘颗粒、重金属、多环芳烃和放射性氡有关，大大增加了患肺癌的风险。本研究旨在探讨在云锡矿粉诱导大鼠肺泡II型上皮细胞（immortalized rat alveolar cells type II, RLE-6TN）恶性转化过程中，瘦素（leptin）及其介导的细胞外调节蛋白激酶（extracellular regulated protein kinase, ERK）信号通路所起的作用。 方法 采用200 μg/mL的云锡矿粉隔代毒染RLE-6TN至第9代，建立毒染细胞模型，命名为R200细胞，正常培养组命名为R细胞，通过Western blot法检测两种细胞leptin受体的表达情况。通过MTT法筛选出leptin及丝裂原活化蛋白激酶激酶（mitogen-activated protein kinase kinase, MEK）抑制剂（U0126）对R200细胞的最佳作用浓度。自第20代起，将R组、R200组细胞分别与leptin及MEK抑制剂U0126共培养，对各组细胞的形态改变进行观察，并利用苏木素-伊红（hematoxylin-eosin, HE）染色技术鉴别第40代细胞的形态学差异，通过刀豆凝集素A（concanavalin A, ConA）及锚着独立性生长实验法检测细胞恶性转化情况。通过Western blot法检测leptin作用后上皮细胞ERK信号通路的变化。 结果 ng/mL时，其促增殖效应最为显著，30 μmol/L U0126可抑制毒染细胞R200增殖，与对照组相比具有统计学差异（P<0.05）。自第25代起，leptin诱导的R200组（R200L组）细胞形态发生变化，至第30代出现恶性转化，至第40代时恶性转化特征明显；而R200组细胞及U0126诱导的R200组（R200LU组）细胞则在第40代时才出现恶性转化特征。R200L组细胞凝集速度较R200LU组快，其余各组细胞P30出现凝集，且随ConA浓度增加，细胞凝集速度加快。R200L组细胞自P40可见克隆形成，克隆形成率为2.25‰±0.5‰，R200LU组及R200组未见克隆集落。R200L组细胞pERK表达增强；加入U0126阻断后，R200L组细胞pERK磷酸化水平降低。 结论 Leptin可以促进云锡矿粉毒染肺上皮细胞的恶性转化，ERK信号通路可能是其促进云锡矿粉引发的肺泡II型上皮细胞转化的重要途径。

sion of leptin receptor in both cell groups was detected using the Western blot method.The optimal concentration of leptin and mitogenactivated protein kinase kinase (MEK) inhibitor (U0126) on R 200 cells was determined using the MTT method.Starting from the 20 th generation, the cells in the R group were co-cultured with leptin, while the cells in the R 200 group were co-cultured with the MEK inhibitor U0126.The morphological alterations of the cells in each group were visualized utilizing hematoxylin-eosin staining.Additionally, concanavalin A (ConA) was utilized to detect any morphological differences, and an anchorage-independent growth assay was conducted to assess the malignant transformation of the cells.The changes in the ERK signaling pathway in epithelial cells after the action of leptin were detected using the Western blot method.Results Both the cells in the R group and R 200 group express leptin receptor OB-R.Compared to the R 200 group, the concentration of leptin at 100 ng/mL shows the most significant pro-proliferation effect.The proliferation of R 200 cells infected with the virus is inhibited by 30 μmol/L U0126, and a statistically significant divergence was seen when compared to the control group (P<0.05).Starting from the 25 th

云南个旧锡矿工人群体被视为肺癌高发群体之一，鳞 癌是其主要的病理组织学类型，且癌灶及癌旁肺组织常伴 发弥漫性纤维化
189 μm [7] ；R LE-6TN细胞株(美国 ATCC公司)；DMEM/F12培养液(美国Hyclone)；细胞   体外增殖 [10,11] ，还能降低癌细胞的凋亡能力 [12] 。 食管癌、 卵巢癌、肺癌、前列腺癌、乳腺癌、大肠癌、 胃癌等恶性肿 瘤中leptin也呈现高表达 [13][14][15][16][17][18][19] ，且血清leptin水平的检测可 中国肺癌杂志 www.lungca.org能提示预后信息。 由于其独特的地域特性，云锡肺癌病理组织学类型 主要表现为鳞状细胞癌，且常伴随肺组织广泛的纤维化 和肿瘤间质反应 [20] 。肺癌与肺纤维化之间存在密切关系， 多数观点认为弥漫性肺纤维化可增加患肺癌的风险， 甚至 有观点认为肺纤维化可以作为独立的指标来预测肺癌的 发生， 但其具体的发病机制仍未完全阐明 [21] 。课题组既往 研究 [22]    中国肺癌杂志 www.lungca.org generation, the cell morphology of the leptin-induced R 200 group (R 200 L group) underwent changes, leading to malignant transformation observed at the 30 th generation.The characteristics of malignant transformation became evident by the 40 th generation in the R 200 L group.In contrast, the other groups showed agglutination of P40 cells, and the speed of cell aggregation increased with an increase in ConA concentration.Notably, the R 200 L group exhibited faster cell aggregation compared to the U0126-induced R 200 (R 200 LU) group.Additionally, the cells in the R 200 L group were capable of forming clones starting from P30, with a colony formation rate of 2.25‰±0.5‰.However, no clonal colonies were observed in the R 200 LU group and R 200 group.The expression of phosphorylated extracellular signal-regulated kinase (pERK) was enhanced in cells of the R 200 L group.However, when the cells in the R 200 L group were treated with U0126, a blocking agent, the phosphorylation level of pERK decreased.Conclusion Leptin can promote the malignant transformation of lung epithelial cells infected by mine dust, and the ERK signaling pathway may be necessary for the transformation of alveolar type II epithelial cells induced by Yunnan tin mine dust.【Key words】 Leptin; Alveolar type II epithelial cells; Transformation; ERK signaling pathway 【Copyright statement】Copyright © 2023, Chinese Journal of Lung Cancer.This study was supported by the grants from National Natural Science Foundation of China (No.82360523, No.82060423), Science and Technology Innovation Team for Precision Pathological Diagnosis of Lung Malignant Tumours of Kunming Medical University (No.CXTD202210) and "Famous Doctor Special Project" of Xingdian Talent Programme (No.RLMY20220018) (All to Li BIAN).

Fig 3 Fig 2
Fig 1 The expression of leptin receptor OB-R in R group and R 200 group was detec ted by Western blot.The cells were cultured normally, named as R cells.RLE-6TN cells were infected with Yunnan tin mine dust at a concentration of 200 μg/mL for nine consecutive generations to establish the infected cell model, which was named R 200 cells.

Fig 5
Fig 5 Clone formation after the action of rRtLeptin and U0126 on R 200 group cells (magnification ×100)

Fig 4
Fig 4 Plot of HE results of rRtLeptin on the morphology of R 200 group cells.HE: hematoxylin-eosin.